Increased IgD and CD27 Double Negative (DN) B cell population in pediatric onset autoimmune hepatitis.

Autoimmune hepatitis (AIH) is a chronic, inflammatory liver disease of unknown aetiology which requires lifelong immunosuppression. Most therapeutic and outcome studies of AIH have been conducted predominantly in Caucasian (European Ancestry, EA) cohorts, with the exclusion of African American (AA) patients due to inadequate sample size. It is known that AA patients have a severe phenotype of autoimmune diseases and demonstrate a poor response to conventional medical therapy. Understanding cellular and molecular pathways which determine AIH severity and progression in AA patients is likely to lead to the discovery of novel, personalised and better tolerated therapies. The aim of the study is to determine the distinct effector B cell phenotypes which contribute to disease severity and progression of AIH in AA children as compared to their EA cohorts. PBMCs were isolated from blood samples collected from patients visiting Children's Healthcare of Atlanta (CHOA) and were grouped into AA, (n = 12), EA, (n = 11) and controls (n = 12) and were processed for flow cytometry. Markers of B cell development, maturation and activation were assessed namely CD19, CD21, IgD, CD27, CD38, CD11c, CD24, CD138. AA children with AIH demonstrated an expansion of CD19 + ve, Activated Naïve (aN), (CD19+ IgD-/CD27- Double Negative (DN2) ([CD19+/IgD-/CD27++CD38++) cells. Plasmablasts were significantly higher along with Signalling Lymphocytic activation molecule F7 (SLAMF7). Unswitched memory [CD19+] IgD+CD27+ (USM) B cells were significantly contracted in AA patients with AIH. B cell phenotyping reveals a distinct profile in AA AIH patients with a major skewing towards the expansion of effector pathways which have been previously characterised in severe SLE in AA patients. These results suggest that the quantification and therapeutic target of B cell pathway could contribute substantially to the clinical approach to AIH especially in the AA population.

Characterization of Intestinal Mesenchymal Stromal Cells From Patients With Inflammatory Bowel Disease for Autologous Cell Therapy.

Therapy with mesenchymal stromal cells (MSCs) has shown promise in inflammatory bowel disease-leveraging their immunosuppressive and regenerative properties. However, the potential immunogenic complications of allogenic MSCs sourced from different tissues raise concern. Thus, we assessed the fitness and functionality of autologous intestinal MSCs as a potential platform for cellular therapy. Mucosal biopsy-derived MSCs from Crohn's disease (n = 11), ulcerative colitis (n = 12), and controls (n = 14) were analyzed by microscopy and flow cytometry for doubling-time, morphology, differentiation potential, and immunophenotype. Gene expression, cell-subtype composition, along with surface marker and secretome changes after IFN-γ priming were measured by bulk and single-cell RNA sequencing coupled with a 30-plex Luminex panel. MSCs expanded ex vivo demonstrate canonical MSC markers, similar growth kinetics, and tripotency regardless of the patient phenotype. Global transcription patterns were similar at baseline though inflammatory bowel disease (IBD) rectal MSCs showed changes in select immunomodulatory genes. IFN-γ priming resulted in upregulation of shared immunoregulatory genes (particularly in PD-1 signaling) and overrode the transcriptional differences observed at baseline. Furthermore, MSCs secrete key immunomodulatory molecules at baseline and in response to IFN-γ including CXCL10, CXCL9, and MCP-1. Overall, MSCs from IBD patients have normal transcriptional and immunomodulatory properties with therapeutic potential and can be sufficiently expanded.

Assessing Cellular and Transcriptional Diversity of Ileal Mucosa Among Treatment-Naïve and Treated Crohn's Disease.

BACKGROUND: Crohn's disease is a lifelong disease characterized by chronic inflammation of the gastrointestinal tract. Defining the cellular and transcriptional composition of the mucosa at different stages of disease progression is needed for personalized therapy in Crohn's. METHODS: Ileal biopsies were obtained from (1) control subjects (n = 6), (2) treatment-naïve patients (n = 7), and (3) established (n = 14) Crohn's patients along with remission (n = 3) and refractory (n = 11) treatment groups. The biopsies processed using 10x Genomics single cell 5' yielded 139 906 cells. Gene expression count matrices of all samples were analyzed by reciprocal principal component integration, followed by clustering analysis. Manual annotations of the clusters were performed using canonical gene markers. Cell type proportions, differential expression analysis, and gene ontology enrichment were carried out for each cell type. RESULTS: We identified 3 cellular compartments with 9 epithelial, 1 stromal, and 5 immune cell subtypes. We observed differences in the cellular composition between control, treatment-naïve, and established groups, with the significant changes in the epithelial subtypes of the treatment-naïve patients, including microfold, tuft, goblet, enterocyte,s and BEST4+ cells. Surprisingly, fewer changes in the composition of the immune compartment were observed; however, gene expression in the epithelial and immune compartment was different between Crohn's phenotypes, indicating changes in cellular activity. CONCLUSIONS: Our study identified cellular and transcriptional signatures associated with treatment-naïve Crohn's disease that collectively point to dysfunction of the intestinal barrier with an increase in inflammatory cellular activity. Our analysis also highlights the heterogeneity among patients within the same disease phenotype, shining a new light on personalized treatment responses and strategies.

Ileal Derived Organoids From Crohn's Disease Patients Show Unique Transcriptomic and Secretomic Signatures.

BACKGROUND & AIMS: We used patient-derived organoids (PDOs) to study the epithelial-specific transcriptional and secretome signatures of the ileum during Crohn's disease (CD) with varying phenotypes to screen for disease profiles and potential druggable targets. METHODS: RNA sequencing was performed on isolated intestinal crypts and 3-week-old PDOs derived from ileal biopsies of CD patients (n = 8 B1, inflammatory; n = 8 B2, stricturing disease) and non-inflammatory bowel disease (IBD) controls (n = 13). Differentially expressed (DE) genes were identified by comparing CD vs control, B1 vs B2, and inflamed vs non-inflamed. DE genes were used for computational screening to find candidate small molecules that could potentially reverse B1and B2 gene signatures. The secretome of a second cohort (n = 6 non-IBD controls, n = 7 CD, 5 non-inflamed, 2 inflamed) was tested by Luminex using cultured organoid conditioned medium. RESULTS: We found 90% similarity in both the identity and abundance of protein coding genes between PDOs and intestinal crypts (15,554 transcripts of 19,900 genes). DE analysis identified 814 genes among disease group (CD vs non-IBD control), 470 genes different between the CD phenotypes, and 5 false discovery rate correction significant genes between inflamed and non-inflamed CD. The PDOs showed both similarity and diversity in the levels and types of soluble cytokines and growth factors they released. Perturbagen analysis revealed potential candidate compounds to reverse B2 disease phenotype to B1 in PDOs. CONCLUSIONS: PDOs are similar at the transcriptome level with the in vivo epithelium and retain disease-specific gene expression for which we have identified secretome products, druggable targets, and corresponding pharmacologic agents. Targeting the epithelium could reverse a stricturing phenotype and improve outcomes.

A two decade long study of disease progression of de novo and recurrent autoimmune hepatitis in the pediatric population.

Recurrent autoimmune hepatitis (rAIH) occurs in patients who undergo liver transplantation (LT) for AIH and de novo AIH (dAIH) is seen in patients who are transplanted for etiologies other than AIH. Whether these are distinct diseases with a similar phenotype remains understudied. The aim of this study was to identify clinical and immunologic factors affecting outcome in patients with dAIH and rAIH. A retrospective review of 387 LT patients from 1997 to 2014 was carried out, and they were followed until 2018. Patients with rAIH or dAIH were identified based on the pre-transplant diagnosis of AIH (or not) and characteristic histology. Liver biopsies were stained with H&E, B-cell marker CD20, and plasma cell marker CD138. Out of 387 patients, 31 were transplanted for AIH, and 8/31 developed rAIH. Of the remaining 356 patients, eight developed dAIH. Compared to the dAIH group, rAIH occurred in older patients, had an earlier onset in the allograft, and had higher IgG and serum ALT levels. It was most commonly seen in African American (AA) patients (87%). rAIH patients had significantly higher CD20 and CD138 positivity in liver biopsies. In addition, they had increased rejection episodes prior to the onset of recurrence, increased graft loss, and mortality. rAIH is a more aggressive disease, and has a preponderance of B cells and plasma cells in the liver tissue as compared to dAIH. The concurrent association with increased graft loss and patient mortality in rAIH warrants further investigations into B cell-targeted therapies.

Mitigation of autophagy ameliorates hepatocellular damage following ischemia-reperfusion injury in murine steatotic liver.

Ischemia-reperfusion injury (IRI) is a common clinical consequence of hepatic surgery, cardiogenic shock, and liver transplantation. A steatotic liver is particularly vulnerable to IRI, responding with extensive hepatocellular injury. Autophagy, a lysosomal pathway balancing cell survival and cell death, is engaged in IRI, although its role in IRI of a steatotic liver is unclear. The role of autophagy was investigated in high-fat diet (HFD)-fed mice exposed to IRI in vivo and in steatotic hepatocytes exposed to hypoxic IRI (HIRI) in vitro. Two inhibitors of autophagy, 3-methyladenine and bafilomycin A1, protected the steatotic hepatocytes from HIRI. Exendin 4 (Ex4), a glucagon-like peptide 1 analog, also led to suppression of autophagy, as evidenced by decreased autophagy-associated proteins [microtubule-associated protein 1A/1B-light chain 3 (LC3) II, p62, high-mobility group protein B1, beclin-1, and autophagy-related protein 7], reduced hepatocellular damage, and improved mitochondrial structure and function in HFD-fed mice exposed to IRI. Decreased autophagy was further demonstrated by reversal of a punctate pattern of LC3 and decreased autophagic flux after IRI in HFD-fed mice. Under the same conditions, the effects of Ex4 were reversed by the competitive antagonist exendin 9-39. The present study suggests that, in IRI of hepatic steatosis, treatment of hepatocytes with Ex4 mitigates autophagy, ameliorates hepatocellular injury, and preserves mitochondrial integrity. These data suggest that therapies targeting autophagy, by Ex4 treatment in particular, may ameliorate the effects of IRI in highly prevalent steatotic liver.

The glucagon-like peptide-1 receptor agonist Exendin 4 has a protective role in ischemic injury of lean and steatotic liver by inhibiting cell death and stimulating lipolysis.

Nonalcoholic fatty liver disease is an increasingly prevalent spectrum of conditions characterized by excess fat deposition within hepatocytes. Affected hepatocytes are known to be highly susceptible to ischemic insults, responding to injury with increased cell death, and commensurate liver dysfunction. Numerous clinical circumstances lead to hepatic ischemia. Mechanistically, specific means of reducing hepatic vulnerability to ischemia are of increasing clinical importance. In this study, we demonstrate that the glucagon-like peptide-1 receptor agonist Exendin 4 (Ex4) protects hepatocytes from ischemia reperfusion injury by mitigating necrosis and apoptosis. Importantly, this effect is more pronounced in steatotic livers, with significantly reducing cell death and facilitating the initiation of lipolysis. Ex4 treatment leads to increased lipid droplet fission, and phosphorylation of perilipin and hormone sensitive lipase - all hallmarks of lipolysis. Importantly, the protective effects of Ex4 are seen after a short course of perioperative treatment, potentially making this clinically relevant. Thus, we conclude that Ex4 has a role in protecting lean and fatty livers from ischemic injury. The rapidity of the effect and the clinical availability of Ex4 make this an attractive new therapeutic approach for treating fatty livers at the time of an ischemic insult.

A(2B)AR expression in non-immune cells plays an important role in the development of murine colitis.

BACKGROUND: Adenosine, an endogenous purine nucleoside, is involved in several physiological functions. We have previously shown that A(2B)AR plays a pro-inflammatory role during colitis. AIMS: Our goals were to determine if A(2B)AR expression was necessary on immune cells/non-immune cells during colitis and if A(2B)AR was a suitable target for treating intestinal inflammation. METHODS: Wild-type and A(2B)AR knockout mice were utilized in bone marrow transplants to explore the importance of immune/non-immune A(2B)AR expression during the development of colitis. Additionally, a T-cell transfer model of colitis was used in Rag1 knockout or A(2B)AR/RAG1 double knockout recipients. Finally, A(2B)AR small interfering RNA nanoparticles were administered to dextran sodium sulphate-treated mice. RESULTS: Wild-type mice receiving wild-type or knockout bone marrow developed severe colitis after dextran sodium sulphate treatment, whereas colitis was significantly attenuated in knockout mice receiving wild-type or knockout bone marrow. Colitis induced in Rag1 knockout animals was attenuated in A(2B)AR/RAG1 double knockout recipients. Animals receiving nanoparticles exhibited attenuated parameters of colitis severity compared to mice receiving control nanoparticles. CONCLUSIONS: Our results suggest that A(2B)AR on non-immune cells plays an important role for the induction of colitis and targeting A(2B)AR expression during colitis may be useful for alleviating symptoms of intestinal inflammation.

A senescence accelerated mouse model to study aging in the larynx.

OBJECTIVE: Age-related changes in the larynx lead to significant voice impairment and reduced quality of life. There is a need for aged animal models that have practical generation times to study the fundamental changes and new therapeutics for the aging voice. The senescence accelerated prone mouse strain (SAMP) animals experience rapid aging without any experimental manipulation. The main objective of this study was to demonstrate the use of senescence accelerated mice to study aging in the larynx. STUDY DESIGN: Murine model. SETTING: Department of Animal Resources, Emory University. SUBJECTS AND METHODS: Larynges from five senescence accelerated prone mice, five normal aging senescence resistant mice, and five C57BL/6 mice were harvested and processed for paraffin sections. Histomorphometry was performed for assessment of collagen and hyaluronic acid distribution. In addition, frozen laryngeal tissue was harvested for transcriptional and translational assessment of collagen-1, using real-time polymerase chain reaction with specific primers and Western blots. Myofibroblast assessment was performed by immunostaining for the presence of alpha-smooth muscle actin. RESULTS: The deposition of collagen increased at six months of age in the SAMP vocal fold, and the level of collagen-1 mRNA increased with age. The myofibroblast protein alpha-smooth muscle actin was also found at a higher concentration in the SAMP vocal tissue. In contrast, the levels of hyaluronic acid in the vocal folds of SAMP mice decreased with age when compared to age-matched C57BL/6 mice. CONCLUSION: SAMP mice show accelerated, age-related changes in the vocal fold that were evident at as early as six months of age. The use of senescence accelerated mice offers promise as a model to study age-related laryngeal changes.

Adenosine 2B receptor expression is post-transcriptionally regulated by microRNA.

We have reported that epithelial adenosine 2B receptor (A(2B)AR) mRNA and protein are up-regulated in colitis, which we demonstrated to be regulated by tumor necrosis factor alpha (TNF-alpha). Here, we examined the mechanism that governs A(2B)AR expression during colitis. A 1.4-kb sequence of the A(2B)AR promoter was cloned into the pFRL7 luciferase vector. Anti-microRNA (miRNA) was custom-synthesized based on specific miRNA binding sites. The binding of miRNA to the 3'-untranslated region (UTR) of A(2B)AR mRNA was examined by cloning this 3'-UTR downstream of the luciferase gene in pMIR-REPORT. In T84 cells, TNF-alpha induced a 35-fold increase in A(2B)AR mRNA but did not increase promoter activity in luciferase assays. By nuclear run-on assay, no increase in A(2B)AR mRNA following TNF-alpha treatment was observed. Four putative miRNA target sites (miR27a, miR27b, miR128a, miR128b) in the 3'-UTR of the A(2B)AR mRNA were identified in T84 cells and mouse colon. Pretreatment of cells with TNF-alpha reduced the levels of miR27b and miR128a by 60%. Over expression of pre-miR27b and pre-miR128a reduced A(2B)AR levels by >60%. Blockade of miR27b increased A(2B)AR mRNA levels by 6-fold in vitro. miR27b levels declined significantly in colitis-affected tissue in mice in the presence of increased A(2B)AR mRNA. Collectively, these data demonstrate that TNF-alpha-induced A(2B)AR expression in colonic epithelial cells is post-transcriptionally regulated by miR27b and miR128a and show that miR27b influences A(2B)AR expression in murine colitis.

Adenosine 2B receptors (A(2B)AR) on enteric neurons regulate murine distal colonic motility.

Delayed colonic emptying leading to constipation is a significant health concern. We investigated the role of adenosine 2B receptor (A(2B)AR) in modulating distal colonic motility using wild-type and A(2B)AR-knockout (A(2B)AR(-/-)) mice. Colon motility was assessed using stool characteristics and colonic transit. Distal colonic ganglia, isolated by laser capture microdissection, were tested for A(2B)AR expression by RT-PCR. The distal colon contraction and relaxation responses were assessed by electrical field stimulation (EFS) in presence of A(2B)AR agonists, antagonists or inhibitors of nitric oxide (NO) and guanylate cyclase. Nitrite levels were measured in enteric neuronal cultures exposed to A(2B)AR agonists/antagonists. A(2B)AR(-/-) mice display increased stool retention, decreased stool frequency, delayed colonic emptying, and decreased circular muscle relaxation. RT-PCR identified A(2B)AR expression in distal colonic ganglia. EFS studies revealed that enteric neuronal A(2B)AR is essential for distal colonic relaxation, and A(2B)AR antagonists can inhibit relaxation. Enteric neurons stimulated with A(2B)AR agonists produced more nitrite than cultures treated with antagonists. We demonstrate an essential role of A(2B)AR in regulating distal colon relaxation, as A(2B)AR activation is linked to NO signaling. Hence targeting the colonic A(2B)AR could represent a novel therapeutic strategy to treat constipation.

Targeted deletion of neuropeptide Y (NPY) modulates experimental colitis.

BACKGROUND: Neurogenic inflammation plays a major role in the pathogenesis of inflammatory bowel disease (IBD). We examined the role of neuropeptide Y (NPY) and neuronal nitric oxide synthase (nNOS) in modulating colitis. METHODS: Colitis was induced by administration of dextran sodium sulphate (3% DSS) or streptomycin pre-treated Salmonella typhimurium (S.T.) in wild type (WT) and NPY (NPY(-/-)) knockout mice. Colitis was assessed by clinical score, histological score and myeloperoxidase activity. NPY and nNOS expression was assessed by immunostaining. Oxidative stress was assessed by measuring catalase activity, glutathione and nitrite levels. Colonic motility was assessed by isometric muscle recording in WT and DSS-treated mice. RESULTS: DSS/S.T. induced an increase in enteric neuronal NPY and nNOS expression in WT mice. WT mice were more susceptible to inflammation compared to NPY(-/-) as indicated by higher clinical & histological scores, and myeloperoxidase (MPO) activity (p<0.01). DSS-WT mice had increased nitrite, decreased glutathione (GSH) levels and increased catalase activity indicating more oxidative stress. The lower histological scores, MPO and chemokine KC in S.T.-treated nNOS(-/-) and NPY(-/-)/nNOS(-/-) mice supported the finding that loss of NPY-induced nNOS attenuated inflammation. The inflammation resulted in chronic impairment of colonic motility in DSS-WT mice. NPY -treated rat enteric neurons in vitro exhibited increased nitrite and TNF-alpha production. CONCLUSIONS: NPY mediated increase in nNOS is a determinant of oxidative stress and subsequent inflammation. Our study highlights the role of neuronal NPY and nNOS as mediators of inflammatory processes in IBD.

A2B adenosine receptor gene deletion attenuates murine colitis.

BACKGROUND & AIMS: The A(2B) adenosine receptor (A(2B)AR) is the predominant adenosine receptor expressed in the colonic epithelia. We have previously shown that A(2B)AR mRNA and protein levels are up-regulated during colitis. In this study, we addressed the role of the A(2B)AR in the development of murine colitis and the potential mechanism underlying its effects. METHODS: Dextran sodium sulfate (DSS), 2,4,6-trinitrobenzene sulfonic acid (TNBS), and Salmonella typhimurium were used to induce colitis in A(2B)AR-null mice (A(2B)AR(-/-)). Colitis was determined using established clinical and histologic scoring. Keratinocyte-derived chemokine (KC) measurements were performed using an enzyme-linked immunosorbent assay. RESULTS: Colonic inflammation induced by DSS, TNBS, or S typhimurium was attenuated in A(2B)AR(-/-) compared with their wild-type counterparts. Clinical features, histologic score, and myeloperoxidase activity were significantly decreased in A(2B)AR(-/-) mice. However, A(2B)AR(-/-) showed increased susceptibility to systemic Salmonella infection. Tissue levels of the neutrophil chemokine, KC was decreased in colitic A(2B)AR(-/-) mice. In addition, flagellin-induced KC levels were attenuated in A(2B)AR(-/-) mice. Neutrophil chemotaxis in response to exogenous interleukin-8 was preserved in A(2B)AR(-/-) mice, suggesting intact neutrophil migration in response to appropriate stimuli. CONCLUSIONS: These data demonstrate, for the first time, that the A(2B)AR plays a proinflammatory role in colitis. A(2B) receptor antagonism may be an effective treatment for acute inflammatory intestinal diseases such as acute flare of inflammatory bowel disease.

Purinergic receptors in gastrointestinal inflammation.

Purinergic receptors comprise a family of transmembrane receptors that are activated by extracellular nucleosides and nucleotides. The two major classes of purinergic receptors, P1 and P2, are expressed widely in the gastrointestinal tract as well as immune cells. The purinergic receptors serve a variety of functions from acting as neurotransmitters, to autocoid and paracrine signaling, to cell activation and immune response. Nucleosides and nucleotide agonist of purinergic receptors are released by many cell types in response to specific physiological signals, and their levels are increased during inflammation. In the past decade, the advent of genetic knockout mice and the development of highly potent and selective agonists and antagonists for the purinergic receptors have significantly advanced the understanding of purinergic receptor signaling in health and inflammation. In fact, agonist/antagonists of purinergic receptors are emerging as therapeutic modalities to treat intestinal inflammation. In this article, the distribution of the purinergic receptors in the gastrointestinal tract and their physiological and pathophysiological role in intestinal inflammation will be reviewed.

Evidence for a sequential transfer of iron amongst ferritin, transferrin and transferrin receptor during duodenal absorption of iron in rat and human.

AIM: To elucidate the sequential transfer of iron amongst ferritin, transferrin and transferrin receptor under various iron status conditions. METHODS: Incorporation of 59Fe into mucosal and luminal proteins was carried out in control WKY rats. The sequential transfer of iron amongst ferritin, transferrin and transferrin receptor was carried out in iron deficient, control and iron overloaded rats. The duodenal proteins were subjected to immunoprecipitation and quantitation by specific ELISA and in situ localization by microautoradiography and immunohistochemistry in tandem duodenal sections. Human duodenal biopsy (n = 36) collected from subjects with differing iron status were also stained for these proteins. RESULTS: Ferritin was identified as the major protein that incorporated iron in a time-dependent manner in the duodenal mucosa. The concentration of mucosal ferritin was significantly higher in the iron excess group compared to control, iron deficient groups (731.5 +/- 191.96 vs 308.3 +/- 123.36, 731.5 +/- 191.96 vs 256.0 +/- 1.19, P < 0.005), while that of luminal transferrin which was significantly higher than the mucosal did not differ among the groups (10.9 +/- 7.6 vs 0.87 +/- 0.79, 11.1 +/- 10.3 vs 0.80 +/- 1.20, 6.8 +/- 4.7 vs 0.61 +/- 0.63, P < 0.001). In situ grading of proteins and iron, and their superimposition, suggested the occurrence of a sequential transfer of iron. This was demonstrated to occur through the initial binding of iron to luminal transferrin then to absorptive cell surface transferrin receptors. The staining intensity of these proteins varied according to the iron nutrition in humans, with intense staining of transferrin receptor observed in iron deficient subjects. CONCLUSION: It is concluded that the intestine takes up iron through a sequential transfer involving interaction of luminal transferrin, transferrin-transferrin receptor and ferritin.

Adenosine 2b receptor (A2bR) signals through adenylate cyclase (AC) 6 isoform in the intestinal epithelial cells.

Adenosine 2b receptor (A2bR), a G-protein coupled receptor positively coupled to adenylate cyclase, mediates key events such as chloride, IL-6 and fibronectin secretion in intestinal epithelial cells and is upregulated during intestinal inflammation. In order to gain insight into the overall mechanism of A2bR activation, in this study, we sought to characterize the AC isoform associated with A2bR signaling. The colonic epithelial cell line T84, expressing only the A2b subtype of adenosine receptor, and Chinese hamster ovary (CHO) cells, were used in these studies. cAMP was measured by luminometric assay and AC isoform expression was determined by Western blot, RT-PCR, isoform-specific stealth RNAi and Quantigene. T84 and CHO cells express all nine known AC isoforms. In order to characterize which AC isoform(s) are associated with A2bR, we used the differential inhibition of specific AC isoforms by calcium and nitric oxide. Pretreatment of cells with carbachol or nitric oxide donors such as S-Nitroso-N-acetylpencillamine (SNAP) and PAPANANOATE inhibited A2bR mediated increase in cAMP. Further, overexpression of AC-5 or AC-6 potentiated A2bR-mediated increases in cAMP levels. Finally, transfection with AC isoform-specific RNAi demonstrated that AC-6 but not AC-5 RNAi inhibited adenosine-induced cAMP levels. Taken together, these results suggest that A2bR mediates signaling through AC-6 isoform. Since pro-inflammatory cytokines such as interferon-gamma (IFN-gamma) modulate the expression of specific AC isoforms in the intestinal epithelia, our observation may have therapeutic implications for intestinal inflammation or diarrhea wherein aA2bR is upregulated.

Targeted deletion of metalloproteinase 9 attenuates experimental colitis in mice: central role of epithelial-derived MMP.

BACKGROUND & AIMS: There is mounting evidence that matrix metalloproteinases are the predominant proteinases expressed in the gut mucosa during active inflammatory bowel disease. We investigated the role of metalloproteinase 9 (MMP-9), a secreted gelatinase that is consistently up-regulated in both animal models and human inflammatory bowel disease and is associated with disease severity, in the pathogenesis of colitis by using mice containing a targeted deletion of the MMP-9 gene. METHODS: Dextran sodium sulfate-induced colitis and Salmonella typhimurium-induced enterocolitis were used as animal models to study colitis. RESULTS: MMP-9 activity and protein expression were absent from normal colonic mucosa but were up-regulated during experimental colitis. MMP-9-/- mice exposed to dextran sodium sulfate or salmonella had a significantly reduced extent and severity of colitis. Immunohistochemical studies showed that MMP-9 was localized to epithelial cells and granulocytes during active colitis. The immune response to systemic administration of Salmonella typhimurium was not affected in MMP-9-/- mice. Neutrophil transmigration studies and bone marrow chimeras showed that neutrophil MMP-9 is neither required for its migration nor sufficient to induce tissue damage during colitis and that epithelial MMP-9 is important for tissue damage. MMP-9 inhibited cell attachment and wound healing in the model intestinal epithelial cell line, Caco2-BBE. CONCLUSIONS: Taken together, our data suggest that MMP-9 expressed by epithelial cells may play an important role in the development of colitis by modulating cell-matrix interaction and wound healing. Thus, strategies to inhibit MMP-9 may be of potential therapeutic benefit.

Polarized fibronectin secretion induced by adenosine regulates bacterial-epithelial interaction in human intestinal epithelial cells.

Fibronectin (FN) is a multifunctional protein that plays important roles in many biological processes including cell adhesion and migration, wound healing and inflammation. Cellular FNs are produced by a wide variety of cell types including epithelial cells, which secrete them and often organize them into extensive extracellular matrices at their basal surface. However, regulation of FN synthesis and the polarity of FN secretion by intestinal epithelial cells have not been investigated. In the present study we investigated the role of adenosine, whose levels are up-regulated during inflammation, in modulating FN synthesis, the polarity of FN secretion and the downstream effects of the secreted FN. Polarized monolayers of T84 cells were used as an intestinal epithelial model. Adenosine added to either the apical or basolateral aspect of the cells led to a time- and dose-dependent accumulation of FN in the culture supernatants, polarized to the apical compartment and reached maximal levels 24 h after apical or basolateral addition of adenosine. Confocal microscopy confirmed that FN localized to the apical domain of model intestinal epithelial cells stimulated with apical or basolateral adenosine. The induction of FN was significantly down-regulated in response to the adenosine receptor antagonist alloxazine and was inhibited by cycloheximide. Moreover, adenosine increased FN promoter activity (3.5-fold compared with unstimulated controls) indicating that FN induction is, in part, transcriptionally regulated. Interestingly, we demonstrated that adenosine, as well as apical FN, significantly enhanced the adherence and invasion of Salmonella typhimurium into cultured epithelial cells. In summary, we have shown for the first time that FN, a classic extracellular matrix protein, is secreted into the apical compartment of epithelial cells in response to adenosine. FN may be a critical host factor that modulates adherence and invasion of bacteria, thus playing a key role in mucosal immune responses during inflammation.